In recent years, as a method for detecting a mutation or a polymorphism in a gene, a method for analyzing a melting curve of a double-stranded nucleic acid composed of a target nucleic acid and a probe (a melting curve analysis method) has been employed widely. According to the melting curve analysis method, by analyzing the presence or absence of a peak at a melting temperature (Tm) of the double strand in a melting curve, the determination of a polymorphism in a gene or the detection of the presence or absence of a mutation in a gene becomes possible.
A Tm generally is as defined below. The absorbance at 260 nm increases as a solution containing a double-stranded DNA is heated. This increase is caused by the fact that the hydrogen bond between both the strands in a double-stranded DNA is released by heating, and the double-stranded DNA is dissociated into single-stranded DNAs (melting of DNA). When every double-stranded DNA is dissociated into single-stranded DNAs, the solution exhibits an absorbance about 1.5 times as large as the absorbance at the time when the heating was initiated (the absorbance of the solution containing only the double-stranded DNA), whereby it can be determined that the melting is completed. Based on this phenomenon, a melting temperature Tm (° C.) generally is defined as a temperature at the time when the amount of increase in absorbance reaches 50% of the total amount of increase in absorbance.
By utilizing such nature of a double-stranded DNA, a polymorphism or a mutation in a target site can be detected in the following manner, for example. That is, it can be achieved by the method in which, using a mutant-type detection probe that is complementary to a target nucleic acid sequence containing a mutant type target site, a double-stranded nucleic acid composed of a single-stranded nucleic acid to be analyzed and the probe is formed, the formed double-stranded nucleic acid is heat-treated, the dissociation of the double strand with temperature increase is detected by measuring signal values such as absorbance and the like, and the presence or absence of a mutation in the target site is determined by the behavior of the obtained signal values (ref. Non Patent Citation 1 and Patent Citation 1). The Tm value becomes higher as the homology of a double-stranded nucleic acid becomes higher and becomes lower as the homology of a double-stranded nucleic acid becomes lower. Thus, as evaluation criteria, a Tm value of a double-stranded DNA composed of a target nucleic acid sequence with a mutant-type target site and a mutant-type detection probe that is 100% complementary to the target nucleic acid sequence and a Tm value of a double-stranded DNA composed of a nucleic acid sequence with a wild-type target site and the mutant-type detection probe are determined previously. Since the Tm value becomes higher as the homology of a double-stranded nucleic acid becomes higher as described above, the former, i.e., the Tm value in the case where the target site is of a mutant type (hereinafter, also referred to as “Tmm value”) is relatively high and the latter, i.e., the Tm value in the case where the target site is of a wild type (hereinafter, also referred to as “Tmw value) is relatively low. Subsequently, a melting curve of the double-stranded nucleic acid composed of the single-stranded nucleic acid to be analyzed and the mutant-type detection probe is prepared, and whether a peak is present at the previously determined Tmm value or at the previously determined Tmw value is checked. When the peak is present at around the Tmm value, the nucleic acid sequence is a 100% match to the mutant-type detection probe, whereby the single-stranded nucleic acid to be analyzed can be determined as having a mutant-type polymorphism. On the other hand, when the peak is present at around the Tmw value, the nucleic acid sequence is a mismatch to the mutant-type detection probe in a single base, whereby the single-stranded nucleic acid to be analyzed can be determined as having a wild-type polymorphism.    Non Patent Citation 1: Clinical Chemistry, 2000 46 (5): p. 631-635    Patent Citation 1: JP 2005-58107 A